Process for purifying 2&#39;,3&#39;-dideoxynucleosides

ABSTRACT

A biological process for producing a 2&#39;,3&#39;-dideoxynucleoside from 2&#39;,3&#39;-dideoxyuridine is disclosed. The 2&#39;,3&#39;-dideoxynucleoside can be purified readily using a porous nonpolar resin absorbent.

This is a .[.continuation.]. division of application Ser. No.07/191,370, filed May 9, 1988.Iadd., now U.S. Pat. No.4,835,104.Iaddend..

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The present invention relates to processes for producing and purifying2',3'-dideoxynucleosides, and to processes for producing2',3'-dideoxy-2',3'-didehydronucleosides.

2. Discussion of the Background.

2',3'-Dideoxynucleosides and 2',3'-dideoxy-2',3'-didehydronucleosidesdisplay anti-viral activity but are prohibitively expensive to prepareindustrially. For example, 2',3'-dideoxy-2',3'-didehydronucleosides ofthe formula (I): ##STR1## per se and 2',3'-dideoxynucleosides of theformula (II): ##STR2## which can be obtained by reducing2',3'-dideoxy-2',3'-didehydronucleosides, both display anti-viralactivity and can be utilized, for example, in the treatment of AIDS.

Didehydronucleosides can therefore be used either as drugs or asintermediates for the production of drugs useful to combat viruses (cf,Published Unexamined Japanese Patent Application No. 280500/86 and J.Med Chem., 30, 440 (1987)). (The definition of substituent B is providedinfra.)

Dideoxynucleosides, such as 2',3'-dideoxyadenosine [9-β-D-(2',3'-dideoxyribofranosyl)adenine]; 2',3'-dideoxyinosine [9-β-D-(2',3'-dideoxyribofranosyl)hypoxanthine]; and 2',3'-dideoxyguanosine[9-β- D-([2',3'dideoxyribofranosyl)guanine], also possess powerfulanti-viral activity. All of these materials are therefore expected to beuseful as antiviral medicines, particularly as drugs for the treatmentof AIDS which is an intractable disease of worldwide concern.

2',3'-Dideoxy-2',3'-didehydronucleosides can be provided by a methodwhich uses ribonucleosides as raw material (cf. J. Org. Chem., 39(1974). Another method uses 2'-deoxyribonucleosides as raw materials(cf. J. Amer. Chem. Soc. 88, 1549 (1966) and J. Org. Chem., 32, 817(1967)), etc.

The conventional method for producing 2',3'-dideoxynucleosides is bychemical deoxygenation of nucleosides at the 2' or 3' position, asdescribed in Chem. Pharm. Bull., 22, 128 (1974). But reports on thismethod are very few however, and no industrial manufacturing process hasyet been established on the basis of this basic method. The reasons forthis are due to the facts that in this method (1) protective groups mustbe introduced prior to deoxygenation, (2) the reaction does not proceedsmoothly because of the steric hindrance at the 2'- and 3'-positions,and (3) severe reaction conditions or powerful reagents cannot be usedbecause nucelosides are unstable under such severe conditions. Nothingis so far known about a microbial process for producing2',3'-dideoxynucleosides.

In all of these methods many steps are required and expensive materialsmust be used as the raw materials. The conventional chemical methodsalso have the problems that long reaction steps are involved and theproduct yield is low. These methods are consequently not advantageousfrom an industrial viewpoint. And hence, there has been a need for a newprocess for producing efficiently and inexpensively2',3'-dideoxynucleosides in high yields.

There also has been a need for a method which can provide2',3'-dideoxy-2',3'-didehydronucleosides industrially, efficiently andinexpensively using readily available starting materials.

In an available process for producing 2',3'-dideoxyinosine (DDI), theoxygen atom at the 2'- or 3'-position of the nucleoside is eliminated(see Chem. Pharm. BulL., 22, 128 (1974)). However, this process has notbeen used widely because (1) protective groups must be introduced priorto the reaction, and (2) the deoxygenation reaction tends to be hinderedby severe steric hindrance at the 2'- and 3'-positions.

In cases where DDI is produced from microbial or enzymatic action on asubstrate such as 2',3'-dideoxyuridine (DDU) or2,3-dideoxyribose-1-phosphoric acid, the reaction mixture obtainedcontains, in addition to the desired product (DDI), unreacted DDU,hypoxanthine (Hyp), uracil (Ura) formed by the decomposition of thesubstrate, and small quantities of nucleic acids formed as by-products.

Known purification treatments, such as concentration andrecrystallization, are not suited for obtaining efficiently high purityDDI from these mixtures. This is because impurities, such as Ura, Hyp,etc., have solubilities lower than that of the desired DDI and,consequently, the crystals of DDI which as formed are contaminated withthe impurities. This makes the purification of DDI exceedinglydifficult.

In addition to this, DDI is susceptible to hydrolysis under eitheracidic or neutral conditions. It is therefore difficult to purify DDI bymeans of ion exchange treatment since an acid is utilized for thistreatment and, DDI is consequently hydrolyzed into a 2,3-dideoxyriboseresidue and a hypoxanthine residue.

For the above reasons, the purification and isolation of DDI has beenpracticed only in laboratories by means of repeated liquidcyromatography or thin layer chromatography. No commercial process forthe purification of DDI is available. There has therefore been a needfor an industrially advantageous purification process which makes itpossible to efficiently and inexpensively purify DDI.

Another process has been reported in which 2',3'-dideoxyadenosine (DDA)is subjected to enzymatic deamination (see Biochim. Biophs. Acta.,566(2), 259 (1979). However, this deamination process, too, has not beenpracticed very often due to the reasons noted above.

Due to these difficulties, the isolation and purification of DDA hasbeen practiced only in laboratories by means of repeated liquidchromatography. No commercial process for the purification of DDA isavailable.

In cases where DDA is produced either microbially or enzymatically fromsubstrates such as 2, 3,-dideoxyuridiine (DDU) or2,3-dideoxyribose-1-phosphoric acid, the reaction mixture obtainedcontains, in addition to the desired product (DDA), unreactedsubstrates, i.e., DDU and adenine ("Ad"), uracil ("U") formed by thedecomposition of the substrate, and small quantities of nucleic acidsformed as by-products.

Known treatments, such as concentration and recrystallization, are alsonot suited to obtain highly pure DDA efficiently. This is because bothDDU and DDA have a high solubility and therefore could not be separatedeasily although Ad and U, solubilities of which are relatively small,can be removed by concentration to some extent.

In addition to this, DDA tends to be hydrolyzed under acidic conditions.It is therefore difficult to purify DDA using an ion exchange treatmentsince an acid is needed for elution of DDA and DDA is consequentlyhydrolyzed to a 2,3-dideoxyribose residue and an adenine residue.

In view of the advantageous properties of these materials there is thusa strongly felt need for both a more efficient process for theirproduction and for an effective method for purifying the same.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide a process forthe facile production of 2',3'-dideoxynucleosides.

It is another object of this invention to provide a facile process forthe production of 2',3'-dideoxy-2',3'-didehydronucleosides.

It is another object of this invention to provide a process for theeconomical production of 2',3'-dideoxynucleosides.

It is another object of this invention to provide a process for theeconomical production of 2',3'-dideoxy-2',3'-didehydronucleosides.

It is another object of this invention to provide a process for thefacile purification of 2',3'-dideoxynucleosides produced in a microbialprocess.

It is another object of this invention to provide a process for thepurification of 2',3'-dideoxynucleosides produced in an enzymaticprocess.

The present invention thus provides processes which satisfy all of theabove object of the invention and others which will become apparent fromthe description of the invention given hereinbelow.

In the process of producing 2',3'-dideoxynucleosides in accordance withthe present invention, one can start from 2',3'-dideoxyuridine. The2',3'-dideoxyuridine, phosphoric acid or a salt thereof, and amicroorganism are combined in an aqueous medium. The microorganism used(1) belongs to one of the following genera: Escherichia, Flavobacterium,Serratia, Enterobacter, Erwinia, Citrobacter, Corynebacterium, Hafina,Kluyvera, Sarmonella or Xanthomonas, and (2) is capable of producing2,3-dideoxyribose-1-phosphate from 2',3'-dideoxyuridine and phosphoricacid or a salt thereof. This first step produces 2,3-dideoxyribose1-phosphate.

This 2,3-dideoxyribose 1-phosphate, an appropriate base, and amicroorganism are combined in an aqueous medium. The microorganism used(1) belongs to the genera Escherichia, Flavobacterium, Serratia,Enterobacter, Erwinia, Citrobacter, Corynebacterium, Hafnia, Kluyvera,Sarmonella or Xanthomonas, and (2) is capable of producing thecorresponding 2',3'-dideoxynucleoside from 2,3-dideoxyribose 1-phosphateand a base. The base is defined infra. This second step produces thedesired 2',3'-dideoxynucleoside.

As should be clear from the above, in one of its aspects the presentinvention provides a two-component process. In the first component2,3-dideoxyribose 1-phosphate is produced. In the second component a2',3'-dideoxynucleoside is produced.

In another aspect of the present invention, a 2',3'-dideoxyuridine isobtained from uridine. In this aspect of the present invention, uridineis converted into a compound of the formula (III): ##STR3## wherein R¹is a C₁₋₁₂ alkyl group which may be linear, branched or cyclic. Thecompound of the formula (III): ##STR4## is then reacted with an acidanhydride to provide 2',3'-dideoxy-2',3'-didehydrouridine. This2',3'-dideoxy-2',3'-didehydrouridine is then reduced to thecorresponding 2',3'-dideoxyuridine. The 2',3'-dideoxyuridine, phosphoricacid or a salt thereof, a base, and a microorganism are then combined inan aqueous medium to obtain a 2',3'-dideoxynucleoside. The microorganismused (1) belongs to the genera Escherichia, Flavobacterium, Seratia,Enterobacter, Erwinia, Citrobacter, Corynebacterium, Hafnia, Kluyvera,Sarmonella or Xanthomonas, and (2) is capable of producing, from2',3'-dideoxyuridine, phosphoric acid or a salt thereof, and a base, thecorresponding 2',3'-dideoxynucleoside. The base used in this aspect ofthe present invention is defined infra.

Thus it should be clear from the above, that the present invention alsoprovides a process for producing, on the one hand, 2',3'-dideoxyuridinefrom uridine, and, on the other hand, a process for producing a2',3'-dideoxynucleoside from 2',3'-dideoxymuridine.

The present invention also provides a process for the purification of2',3'-dideoxynucleosides, in particular 2',3'-dideoxynucleosidesobtained from a microbial or enzymatic process, or combination thereof.In this purification process, the 2',3'-dideoxynucleoside is firstadsorbed onto a porous nonpolar resin, the 2',3'-dideoxynucleoside isthen isolated in its adsorbed form from unwanted contaminants, and theadsorbed 2',3'-dideoxynucleoside is then eluted from the porous nonpolarresin to obtain a pure product.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In a first embodiment the present invention provides a process forproducing a 2',3'-dideoxynucleoside which comprises (1) contacting (1a)a microorganism. (1b) 2,3-dideoxyribose 1-phosphate and (1c) a base inan aqueous medium. The microorganism (i) is at least one member selectedfrom the group consisting of the genera Escherichia, Flavobacterium,Serratia, Enterobacter, Erwinia, Citrobacter, Corynebacterium, Hafnia,Kluyvera, Sarmonella and Xanthomonas, and (ii) is capable of producingthe corresponding 2',3'-dideoxynucleoside from 2,3-dideoxyribose1-phosphate and a base.

In another embodiment of the present invention the 2,3-dideoxyribose1-phosphate is obtained by contacting a microorganism,2',3'-dideoxyuridine and phosphoric acid, or a salt thereof in anaqueous medium. The microorganism used (i) belongs to the genusEscherichia, Flavobacterium, Serratia, Enterobacter, Erwinia,Citrobacter, Corynebacterium, Hafnia, Kluyvera, Sarmonella andXanthomonas, and (ii) is capable of producing 2,3-dideoxyribose1-phosphate from 2',3'-dideoxyuridine and phosphoric acid or a saltthereof.

In another embodiment the present invention provides a process forproducing a 2',3'-dideoxynucleoside by contacting a microorganism,2',3'-dideoxyuridine, phosphoric acid or a salt thereof, and a base inan aqueous medium. The microorganism (i) belongs to the genusEscherichia, Flavobacterium, Serratia, Enterobacter, Erwinia,Citrobacter, Corynebacterium, Hafnia, Kluyvera, Sarmonella andXanthomonas, and (ii) is capable of producing the corresponding2',3'-dideoxynucleoside from 2',3'-dideoxyuridine, phosphoric acid or asalt thereof and a base.

In another embodiment the 2',3'-dideoxyuridine is obtained by (1)converting uridine into a compound of the formula (III) ##STR5## whereinR¹ is a C₁₋₁₂ alkyl group which is linear, branched or cyclic, andreacting the compound of formula (III) with an acid anhydride to obtain2',3'-dideoxy-2,3'-didehydrouridine which is then (3) reduced to obtain2',3'-dideoxyuridine.

As a result of extensive investigations to solve the problem outlined inthe discussion of the background of the invention supra, the presentinventors have found that didehydronucleosides having a basic skeletonof the by formula (IV): ##STR6## can be readily produced in a high yieldby converting compounds having a basic skeleton of the by formula (V):##STR7## in the molecule thereof into compounds having a basic skeletonof the formula (III): ##STR8## in the molecule thereof and then reactingthe compounds of formula (III) with acid anhydrides at a temperature inthe range of, for example, 20° to 200° C. On the basis of this finding,the present inventors have accomplished the present invention. In theformulae above, R¹ represents an alkyl group having 1 to 12 carbonatoms.

The compounds of formula (V) which one used as the starting materials ofthe present invention are, for example, compounds represented by generalformula (VI): ##STR9##

The compounds of formula (III) are, for example, nucleoside derivativesrepresented by formula (VII): ##STR10##

In formulae (VI) and (VII) R¹ represents an alkyl group having 1 to 12carbon atoms. R² represents a hydrogen atom, or an acyl group, anaralkyl group, a silyl group or the like. B represents a base bound to aribose residue, such as a purine base bound at the 9-position thereof ora pyrimidine base bound at the 1-position thereof such as is known innucleic acid chemistry.

The nucleoside derivatives of formula (VII) are reacted with acidanhydrides, for example, at temperatures of from 20° to 200° C., whereby2',3'-dideoxy-2',3'-didehydronucleosides of formula (VIII): ##STR11##are obtained, where R² and B have the same meanings as provided above.

The nucleoside derivatives of formula (VII) can be prepared fromcompounds of formula (VI) using known techniques (cf. Tetrahedron, 23,2301 (1967)).

In the formulae, R¹ represents an alkyl group. Examples of this alkylgroup include methyl group, ethyl group, n-propyl group, etc. R²represents a hydrogen atom or, a C₁₋₆ acyl group, a C₇₋₁₄ aralkyl group,a silyl group or the like. B represents a base bound to a glucoseresidue, such as a purine base bound at the 9-position thereof or apyrimidine base bound at the 1-position thereof.

Examples of the purine base include adenine, quanine, hypoxanthine,xanthine, 6-chloropurine, 6-mercaptopurine, 6-methylthiopurine,2,6-dichloropurine, 2-chloropurine, 2,6-diaminopurine,2-amino-6-chloropurine, 2-aminopurine, etc. Examples of the pyrimidinebase include uracil, cytosine, thymine, 5-fluorouracil, 5-chlorouracil,5-bromouracil, 5-iodouracil, 5-ethyluracil, 5-orotic acid, etc. Inaddition, other bases which can also be used include5-amino-4-imidazolecarbonamide, 1,2,4-triazole-3-carboxamide, etc.

If necessary and/or desired, the amino group in the base moiety may beprotected. The acid anhydrides which can be used in the presentinvention are not particularly limited but from a practical standpoint,anhydrides of fatty acids having 1 to 4 carbon atoms are preferred. Inthe reaction, it is sufficient to use the acid anhydride, but additionalsolvents may also be additionally used. The reaction temperature isbetween 20° and 200° C. The reaction time is 30 minutes to 24 hours.

The progress of the reaction can be tracked by thin layer chromatographyor high performance liquid chromatography. After completion of thereaction, the desired didehydronucleosides of formula (VIII) can beisolated in a conventional manner such as by extraction,recrystallization or the like. If necessary, the didehydronucleosides offormula (VIII) can be further converted into2',3'-dideoxy-2',3'-didehydronucleosides of formula (I): ##STR12## byremoving the protective groups, or into 2',3'-dideoxynucleosides offormula (II). ##STR13##

With the present invention didehydronucleosides can be produced in asimple manner and at low cost. Therefore, the present invention isextremely useful for the medical industry.

Intensive studies have led the inventors to find that2',3'-dideoxynucleosides can be efficiently produced by enzymaticreaction .if 2',3'-dideoxyuridine or 2,3-dideoxyribose 1-phosphate(compounds which can be stably supplied at low costs by chemicalsynthesis) are used as the substrate. One embodiment of this inventionwas accomplished on the basis of these findings.

The reactions involved in the process of this invention may beillustrated as shown below: ##STR14##

Thus an embodiment of this invention involves two types of reactions:(1) a process for producing 2',3'-dideoxynucleosides by the action of amicroorganism upon 2,3-dideoxyribose 1-phosphate and a base; and (2) aprocess for directly producing 2',3'-dideoxynucleosides by the action ofa microorganism upon 2',3'-dideoxyuridine, phosphoric acid or a saltthereof, and a base.

For example, the two types of reactions involved in the process of thisinvention are: (1) a process for producing 2',3'-dideoxyadenosine,2',3'-dideoxyinosine or 2',3'-dideoxyguanosine by the action of amicroorganism upon 2,3-dideoxyribose 1-phosphate and adenine,hypoxanthine or guanine; and (2) a process for directly producing2',3'-dideoxyadenosine, 2',3'-dideoxyinosine or 2',3'-dideoxyguanosineby the action of a microorganism upon 2',3'-dideoxyuridine, phosphoricacid or a salt thereof, and adenine, hypoxanthine or guanine.

This invention also involves a process for producing 2,3-dideoxyribose1-phosphate (a precursor of 2',3'-dideoxynucleosides) by the action of amicroorganism upon 2',3'-dideoxyuridine and phosphoric acid or a saltthereof.

The microorganisms used in the process of this invention have both theenzymic activity to convert 2',3'-dideoxyuridine and phosphoric acid to2,3-dideoxyribose 1-phosphate, and the enzymic activity to convert2,3-dideoxyribose 1-phosphate and adenine, hypoxanthine or guanine to2',3'-dideoxyadenosine, 2',3'-dideoxyinosine or 2',3'-dideoxyguanosine.Hence any one of these microorganisms can be used in any one of theabove processes.

These are strains which belong to the genus Escherichia, Flavobacterium,Serratia, Enterobacter, Erwinia, Citrobacter, Corynebacterium, Hafnia,Kluyvera, Sarmonella or Xanthomonas. The following may be mentioned asillustrative examples of these microorganisms:

    ______________________________________                                        Escherichia coli     ATCC 10798                                               Flavobacterium rhenanum                                                                            FERM BP-1862                                             Serratia rubefaciencs                                                                              FERM BP-1863                                             Enterobacter aerogenes                                                                             ATCC 13048                                               Erwinia carotovora   FERM BP-1538                                             Citrobacter freundii ATCC 8090                                                Corynebacterium vitarumen                                                                          ATCC 10234                                               Hafnia alvei         ATCC 9760                                                Kluyvera citrophila  FERM P-1349                                              Sarmonella schottmuelleri                                                                          ATCC 8759                                                Xanthomonas citri    FERM BP-1861                                             ______________________________________                                    

2',3'-Dideoxynucleosides, or 2,3-dideoxyribose 1-phosphate which is anintermediate therefor, may be formed by the action of a microorganismslisted above either by the culture method in which microorganism iscultivated in the presence of the substrates, or by the enzyme react ionmethod in which grown cells of said microorganism or a treated productthereof are allowed to act upon the substrates.

In the culture method, a commonly employed medium containing carbonsources, nitrogen sources, inorganic ions such as P, S, Fe and Mn, andas required trace nutrients, such as vitamins, and organic nitrogensources, such as protein decomposition products and yeast extract, isused as a basal medium. When, for example, 2,3-dideoxyribose 1 phosphateis to be produced, 2',3'-dideoxyuridine and phosphoric acid (or a saltthereof) are added to this basal medium.

When producing, for example, 2',3'-dideoxyadenosine,2',3'-dideoxyinosine or 2',3'-dideoxyguanosine from 2,3-dideoxyribose1-phosphate, 2,3-dideoxyribose 1-phosphate and adenine, hypoxanthine orguanine are added the above basal medium.

When, for example, 2',3'-dideoxyadenosine, 2',3'-dideoxyinosine or2',3'-dideoxyguanosine is to be produced directly from2',3'-dideoxyuridine, 2',3'-dideoxyuridine, phosphoric acid (or a saltthereof) and adenine, hypoxanthine or guanine are added to the abovebasal medium.

The substrates may be added to the culture medium either at the start ofor during cultivation.

In the enzyme reaction method, various kinds of enzyme sources my beused. These include a culture solution obtained after cultivation of amicroorganism in a commonly employed medium containing carbon sources,nitrogen sources, inorganic ions such as P, S, Fe and Mn, and asrequired trace nutrients, such as vitamin, and organic nitrogen sources,such as protein decompositions products and yeast extract; microbialcells separated from said culture solution; and a treated productthereof (for example, microbial cells dried by the use of acetone, celldebris, ultrasonicated cells, cells treated with toluene or asurface-active agent, cells treated with an enzyme such as lysozyme,protein fraction isolated from cells by extraction followed bysalting-out and other purification steps, purified protein fractionhaving the activity for the intended enzyme reaction, and immobilizedproducts of the microbial cells and treated products thereof).

The preferable concentration of 2',3'-dideoxyuridine or2,3-dideoxyribose 1-phosphate used as a substrate is in the range from 1to 1000 mM. When producing 2,3-dideoxyribose 1-phosphate, the amount ofphosphoric acid or a salt thereof to be added is at least equimolar tothat of 2',3'-dideoxyuridine (preferably 1 to 10 molar proportions);when producing a 2',3'-dideoxynucleoside, on the other hand, its mountis smaller than the above (preferably 0.01 to 10 molar proportions)because the acid is recycled in the reaction system. Any type ofphosphoric acid salt that does not retard the reaction can be used,illustrative examples including inorganic salts, such as Na, K, NH₄ ' Caand Mg salts, and organic salts such as trimethylammonium salt.

Suitable amount of the base (adenine, hypoxanthine or guanine) to beadded is at least equimolar to that of 2',3'-dideoxyuridine or2,3-dideoxyribose 1-phosphate (preferably 1 to 10 molar proportions),when producing 2',3'- dideoxyadenosine, 2',3'-dideoxyinosine or2',3'-dideoxyguanosine directly from 2',3'-dideoxyuridine and whenproducing 2',3'-dideoxyadenosine , 2',3'-dideoxyinosine or2',3'-dideoxyguanosine from 2,3-dideoxyribose 1-phosphate.

In the direct method starting from 2',3'-dideoxyuridine, however, theamount of base may be less than equimolar, when unreacted substrate(2',3'-dideoxyuridine), if left in the reaction mixture, offers noproblem in the succeeding purification step.

2,3-Dideoxyribose 1-phosphate used as a substrate may be a commerciallyavailable product, a product prepared by chemical synthesis, or aproduct produced from 2',3'-dideoxyuridine by microbial action andisolated from the culture solution (as disclosed in this invention).

To an aqueous solution containing these substrates, are added theaforementioned microbial cells or a treated product thereof, and thereaction is allowed to proceed by holding the mixture at 20° to 70° C.(preferably 40° to 70° C.) controlled pH in the range from 4 to 10, thusaccumulating, in the culture solution, a 2',3'-dideoxynucleoside (finalproduct) or 2,3-dideoxyribose 1-phosphate (intermediate).

The 2',3'-dideoxynucleoside (final product) or 2,3-dideoxyribose1-phosphate (intermediate) can be recovered from the culture solution byknown techniques (for example, by utilizing the difference in solubilityin water and organic solvents, and by the use of ion-exchange andadsorption resins). The amounts of these compounds can be determined byhigh-performance liquid chromatography.

The process of this invention produces 2',3'-dideoxyadenosine,2',3'-dideoxyinosine and 2',3'-dideoxyguanosine at higher yields bysimple operations, as compared to conventional, chemical syntheticmethods.

2',3'-Dideoxyadenosine (DDA), 2',3'-dideoxyinosine (DDI) and2',3'-dideoxyguanosine produced by the process of this invention havepowerful antiviral action, and are therefore expected to be useful asantiviral agents, particularly for the treatment of AIDS which is anintractable disease of worldwide concern.

The inventors have now also found that DDI can be separated from suchimpurities Ura, Hyp, etc. when a crude DDI solution, after beingsubjected, e.g., to a treatment for removing cells, a treatment forremoving proteins and/or a treatment for decolorization, is treated witha porous nonpolar resin, preferably in combination with crystallizationand separation steps.

Accordingly, there is provided in another embodiment of the presentinvention a process for the purification of DDI which is characterizedin that, upon the purification of DDI produced with the action of amicroorganism or an enzyme or purification of crude DDI derivedtherefrom, said DDI is adsorbed on a porous nonpolar resin. Preferablythe adsorption step is carried out in combination with a crystallizationstep.

Any crude DDI can be purified in accordance with the process of theinvention, irrespective of its purity. For example, a solution resultingfrom a reaction where a 2,3-dideoxyribose residue is bonded to ahypoxanthine residue with the action of a microorganism or an enzyme, ora crude purification product thereof, can be purified.

Any microorganisms capable of producing DDI can be utilized in thepresent invention, including those belonging to such genera asEscherichia, Flavobacterium, Serratia, Enterobacter, Erwinia,Citrobacter, Corynebacterium, Hafnia, Fluyvera, Salmonella, Xanthomonas,and the like. There are no particular limitations on the enzymes whichcan be used. Any enzymes contained in the above-mentionedmicroorganisms, as well as other enzymes having the same function, canbe utilized.

Reaction mixtures containing DDI to be treated in accordance with theprocess of the invention may contain any impurities, including DDU, Hyp,Ura and nucleic acids formed as by-products. There are no particularlimitations on the concentration of DDI to be contained in the mixtureto be treated, provided that it is within its solubility.

Porous nonpolar resins usable in the process of the invention includestyrene-divinylbenzene copolymers and derivatives thereof modified,e.g., with halogens so as to increase the specific gravity thereof.Examples of such copolymers include Diaion HP series and SP series(manufactured by Mitsubishi Chemical Industries Co., Ltd.), XAD-4(manufactured by Rohm & Haas Co.) and OC 1031 (manufactured by BayerAG). Any other porous nonpolar resins having properties similar to thosementioned above can be used in the process of the invention. Inparticular, those having a high specific gravity, for example, SP 207(manufactured by Mitsubishi Chemical Industries Co., Ltd.) can beadvantageous in the respect of their operability since such resins donot float at the time a DDI-containing solution is fed.

The contact between a porous nonpolar resin and a DDI-containingsolution can be effected by either a batch method or a column method. Acolumn method can be advantageous in terms of easiness in operation.

There are no particular limitations on the rate of passing solutionsthrough the column. In ordinary cases, a space velocity (SV) of from 0.5to 4.0, in particular, from 1 to 2, can be preferable.

The volume load of the DDI-containing solution to be fed to the columndepends on the concentration of DDI contained in the solution. A resinload of DDI of from 5 to 40 g/1-R, in particular, from 10 to 30 g/1-R,can be preferred with regard to separability and economical efficiency.

The temperature of the solution passed through the column can be in therange of from 10° to 50° C. In this temperature range, there are almostno substantial differences in the separability of DDI and suchimpurities as Hyp and Ura, which are contained in the solution.

With regard to the stability of DDI, the pH of the solution to be fed tothe resin is preferably on the alkaline side, in particular, in therange of from 8.0 to 10.0. A temperature not higher than 50° C. can alsobe preferable in respect of its stability.

Explanation will hereinafter be given on the method for eluting DDI fromthe column. An aqueous solution of a lower fatty alcohol can be suitedas an eluent. For example, there can be used aqueous solutions of methylalcohol, ethyl alcohol, isopropyl alcohol or the like. The elution canbe carried out at ordinary space velocity (SV), e.g., of from 1to 2.

The purification procedure utilizing a porous nonpolar resin can bepracticed as follows. A predetermined amount of a DDI-containingsolution is fed to a column charged with a porous nonpolar resin, andUra and Hyp are eluted by passing water through the column. DDI and DDUare then eluted by an aqueous alcohol solution.

Thereafter, the fraction that contains DDI and DDU are concentrated andthen cooled, so as to separate DDI from DDU through crystallization. Ahighly pure DDI can be obtained in this manner.

If the pH of the concentrated solution is maintained on the alkalineside, preferably, in the range of from 8 to 10, the decomposition of theDDI can be prevented at the time of crystallization and, hence, theyield of crystallization can be improved.

If desired, other treatments, such as solvent extraction and liquidchromatography, can be applied thereto, in addition to the treatmentsdescribed hereinabove.

As described hereinabove, DDI can be separated and purified in aneffective manner by the treatment with a porous nonpolar resin accordingto the invention, preferably in combination with crystallization. It istherefore highly expected that the process be industrially practiced.

The inventors have now also found that impurities, such as Ad, U, DDU,etc., can be separated from DDA and that highly pure DDA can be obtainedfrom a DDA-containing fermentation or enzymatic solution, after thatsolution has been subjected, e.g., to a treatment for removing cells, atreatment for removing proteins and/or a treatment for decoloration, hasbeen concentrated and filtered, and the filtrate is then treated with aporous nonpolar resin. Another embodiment of this invention has beencompleted on the basis of the above finding.

Accordingly, there is provided in an embodiment of the present inventiona process for the purification of DDA which is characterized in that,upon the purification of DDA produced with the action of a microorganismor an enzyme, the DDA is adsorbed by a porous nonpolar resin.

Any crude DDA can be purified in accordance with the process of theinvention, irrespective of its purity. For example, a solution resultingfrom a reaction where a 2,3-dideoxyribose residue is bonded to anadenine residue with the action of a microorganism or an enzyme, or acrude purification product thereof, can be purified.

Any microorganisms capable of producing DDA can be utilized in thepresent invention, including those belonging to such genera asEscherichia, Flavobacterium, Serratia, Enterobacter, Erwinia,Citorobacter, Corynebacterium, Hafnia, Fluyvera, Salmonella,Xanthomonas, and the like. There are no particular limitations on theenzymes which can be used. Any enzymes contained in the above-mentionedmicroorganisms, as well as other enzymes exhibiting the same function,can be utilized.

Concentrated DDA-containing solutions which can be treated in accordancewith the process of the invention may contain any impurities, includingDDU, Ad, U and nucleic acids formed as by-products. There are noparticular limitations on the concentration of the solution to betreated, provided that the DDA is dissolved.

Porous nonpolar resins usable in this embodiment of the inventioninclude styrene-divinylbenzene copolymers and derivatives thereofmodified, e.g., with halogens so as to increase their specific gravity.Examples of such copolymers include Dialon HP series and SP series(manufactured by Mitsubishi Chemical Industries, Ltd.), XAD-4(manufactured by Rohm & Haas Co) and OC 1031 (manufactured by Bayer AG).Any other porous nonpolar resins having properties similar to thosementioned above can be used in this embodiment of the invention. Inparticular, those having a high specific gravity, for example, SP 207(manufactured by Mitsubishi Chemical Industries, Ltd.) can beadvantageous in the respect of their operability since such resins donot float at the time when a concentrated DDA-containing solution isfed.

The contact between a porous nonpolar resin and a concentratedDDA-containing solution can be effected by either a batch method or acolumn method. A column method can be advantageous with regard to theeasiness in operation.

There are no particular limitations on the rate of passing solutionsthrough the column. In ordinary cases, a space velocity (SV) of from 0.5to 4.0, in particular, from 1 to 2, can be preferable.

The volume load of the concentrated DDA-containing solution to be fed tothe column depends on the concentration of DDA contained .in thesolution. A resin load of DDA of from 5 to 40 g/l-R, in particular, from10 to 30 g/l-R, can be preferred with regard to separability andeconomical efficiency.

The temperature of the solution to be passed through the column can bein the range of from 10° to 50° C. In this temperature range, there arealmost no substantial differences in the separability of DDA and suchimpurities as Ad, U and DDU, which my be contained in the concentratedsolution.

In the method for eluting DDA from the column, an aqueous solution of alower fatty alcohol can be suitably used as an eluent. For example,there can be used aqueous solutions of methyl alcohol, ethyl alcohol,isopropyl alcohol or the like. The elution can be carried out atordinary space velocity (SV), e.g., of from 1 to 2.

The actual purification procedure utilizing porous nonpolar resin can bepracticed as follows. A predetermined amount of a concentratedDDA-containing solution is fed to a column charged with a porousnonpolar resin, and U is eluted by passing water through the column.Thereafter, DDU and Ad are eluted by an aqueous alcohol solution, andDDA is then eluted by passing an aqueous alcohol solution having ahigher-alcohol concentration. The filtrate that contains DDA isconcentrated and then cooled, so as to obtain highly pure DDA.

Other features of this invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLE 1: Production of 2',3'-dideoxy-2',3'-didehydrouridine

To 50 ml of acetic anhydride was added 5.00 g (17.5 mmols) of2',3'-o-methoxymethylideneuridine at room temperature with stirring. Thesolution was heated to 140° C. and kept at this temperature for 5 hoursunder reflux of the solvent. After cooling to room temperature, thesolvent was removed by distillation under reduced pressure, 50 ml ofwater was added, and the mixture was extracted 3 times with 100 ml ofchloroform. The extract was concentrated under reduced pressure, 30%ammonia water was added and the resultant mixture was stirred at roomtemperature for an hour. The solvent was again removed by distillationunder reduced pressure and purification was performed by silica gelcolumn chromatography to give 3.02 g 14.4 mmols) of the title compound(yield 82.3%) mp 150.0°-151.0° C.; λ_(max) (MeOH) 260 nm (ε9980),λ_(min) (MeOH) 231 m (ε3980); ¹ H-NMR (Me₂ SO-d₆) δ 3.58 (d, 1H, J═5.13Hz), 3.59 (d, 1H, J═5.13 Hz), 4.78 (m, 1H), 4.98 (t, 1H, J═5.13 Hz),5.59 (d, 1H, J═8.05 Hz), 5.92 (d, 1H, J═5.86 Hz), 6.40 (d, 1H, J═5.86Hz), 6.82 (m, 1H), 7.75 (d, 1H, J═8.05 Hz), 11.31 (brs, 1H); Fast atombombardment mass spectrum m/z 211 (MH+). Anal. Calcd for C₉ H₁₀ N₂ O4:C, 51.43; H, 4.80; N, 13.33. Found: C, 51.32; H, 4.81; N, 13.31.

REFERENCE EXAMPLE 1: Production of 2',3'-o-methoxymethylideneuridine

To 1 liter of tetrahydrofuran were added 50 g (0.205 mols) of uridine,112 ml (1.03 mols) of methyl orthoformate and 10 g (52.6 mmols) ofparatoluenesulfonic acid at room temperature with stirring. Afterstirring at room temperature for 24 hours, the reaction mixture waspoured into an aqueous sodium bicarbonate solution followed byextraction with chloroform 5 times. The extract was dried over sodiumsulfate and concentrated to give 49.5 g (0.173 mmols) of2',3'-o-methoxymethylideneuridine (yield, 84.5%).

REFERENCE EXAMPLE 2: Production of 2',3'-dideoxyuridine

A solution of 2',3'-dideoxy-2',3'-didehydrouridine (1.0 g, 4.8 mmol) inmethanol (10 ml) containing a catalyst (wet 5% palladium on carbon) (400mg) was stirred in an atmosphere of hydrogen for 1 h. The catalyst wasremoved by filtration and the filtrate was concentrated. The residue waschromatographed on silica gel (CHCl₃ /MeOH═5/1) to give2',3'-dideoxyuridine (0.99 g, 97% yield): Mp 121.2°-121.7° C.; λ_(max)(MeOH) 262 nm (ε10560), λ_(min) (MeOH) 232 nm (ε3810); ¹ H-NMR (Me₂O-d₆)δ 1.74-2.00 (m, 3H), 2.20-2.35 (m, 1H), 3.49-3.55 (m, 1H),3.64-3.69 (m, 1H), 4.00-4.03 brs, 1H), 5.03 (s, 1H), 5.58 (d, 1H, J═8.06Hz), 5.95 (m, 1H), 7.94 (d, 1H, J═8.06 Hz), 11.25 (brs, 1H); Fast atombombardment mass spectrum m/z 213 (MH+). Anal. Calcd for C₉ H₁₂ N₂ O₄ :C, 50.94; H, 5.70; N, 13.20. Found: C, 50.95; H, 5.71; N, 13.20.

EXAMPLE 2: Production of 2',3'-dideoxyadenosine, 2',3'-dideoxyinosineand 2',3'-dideoxyguanosine

Fifty milliliters of a culture medium (pH 7.0), containing 0.5 g/dlyeast extract, 1.0 g/dl peptone, 1.0 g/dl meat extract and 0.5 g/dlNaCl, was placed in 500-m flasks each and sterilized. To each of theflasks, was inoculated a pinch (Aze) of a microorganism listed in Table1, which had previously been grown in a bouillon medium at 30° C. for 16hours, and shake culture was continued at 30° C. for 16 hours. The growncells were separated from the culture solution by centrifugation, washedwith a 0.05 M phosphate buffer (pH 7.2), and centrifuged again.

The washed microbial cells thus obtained were added to 10 ml of a 0.05 MTris buffer (pH 7.2) containing 20 mM 2,3-dideoxyribose 1-phosphate and20mM adenine (or 20mM hypoxanthine, or 20mM guanine) to a concentrationof 5%, and the mixture was held at 60° C. for 24 hours.

The concentration of 2',3'-dideoxyadenosine, 2',3'-dideoxyinosine or2',3'-dideoxyguanosine formed in each of the reaction mixtures wasmeasured by high-performance liquid chromatography.

                  TABLE 1                                                         ______________________________________                                                  2',3'-Dideoxynucleosides formed (mg/dl)                                         2',3'-Dideoxy-                                                                           2',3'-Dideoxy-                                                                           2',3'-Dideoxy-                              Strains     adenosine  inosine    guanosine                                   ______________________________________                                        Escherichia coli                                                                          302        290        285                                         ATCC 10798                                                                    Flavocaterium                                                                             152        148        151                                         rhenanum                                                                      FERM BP-1862                                                                  Serratia rubefaciencs                                                                     81         62         49                                          FERM BP-1863                                                                  Enterobacter                                                                              25         26         27                                          aerogenes                                                                     ATCC 13048                                                                    Erwinia carotovora                                                                        172        191        186                                         FERM BP-1538                                                                  Citrobacter freundii                                                                      43         29         35                                          ATCC 8090                                                                     Corynebacterium                                                                           36         39         42                                          vitarumen                                                                     ATCC 10234                                                                    Hafnia alvei                                                                              41         46         40                                          ATCC 9760                                                                     Kluyvera citrophila                                                                       44         41         43                                          FERM P-3149                                                                   Salmonella  39         40         36                                          schottmuelleri                                                                ATCC 8759                                                                     Xanthomonas citri                                                                         51         48         50                                          FERM BP-1861                                                                  ______________________________________                                    

EXAMPLE 3

Washed cells of microorganisms listed in Table 2, which had been grownand treated.-in the same way as in Example 1, were added to 10 ml of a100mM phosphate buffer (pH 7.0) containing 20mM 2',3'-dideoxyuridine toa concentration of 5%, and the mixture was heated at 60° C. for 24hours. The concentration of 2,3-dideoxyribose 1-phosphate formed in eachof the reaction mixtures was measured by means of high-performanceliquid chromatography. The results are also shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                              2,3-Dideoxyribose 1-                                                          phosphate formed                                        Strain                (mg/dl)                                                 ______________________________________                                        Escherichia coli ATCC 10798                                                                         108                                                     Flavocaterium rhenanum FERM BP-1862                                                                 72                                                      Serratia rubefaciencs FERM BP-1863                                                                  23                                                      Enterobacter aerogenes ATCC 13048                                                                   18                                                      Erwinia carotovora FERM BP-1538                                                                     91                                                      Citrobacter freundii l ATCC 8090                                                                    47                                                      Corynebacterium vitarumen ATCC 10234                                                                36                                                      Hafnia alvei ATCC 9760                                                                              29                                                      Kluyvera citrophila FERM P-3149                                                                     45                                                      Sarmonella schottmuelleri ATCC 8759                                                                 28                                                      Xanthomonas citri FERM BP-1861                                                                      37                                                      ______________________________________                                    

EXAMPLE 4

Washed cells of microorganisms listed in Table 3, which had been grownand treated in the same way as in Example 2, were added to 10 ml of a100 mM phosphate buffer (pH 7.0) containing 20 mM, 2,3-dideoxyribose1-phosphate and 20 mM adenine (or 20 mM hypoxanthine, or 20 mM guanine)to a concentration of 5%, and the mixture was held at 60° C. for 24hours. The concentration of 2',3'-dideoxynucleoside formed in each ofthe reaction mixtures was measured by means of high-performance liquidchromatography. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                  2',3'-Dideoxynucleosides formed (mg/dl)                                         2',3'-Dideoxy-                                                                           2',3'-Dideoxy-                                                                           2',3'-Dideoxy-                              Strains     adenosine  inosine    guanosine                                   ______________________________________                                        Escherichia coli                                                                          239        202        213                                         ATCC 10798                                                                    Flavocaterium                                                                             131        138        130                                         rhenanum                                                                      FERM BP-1862                                                                  Serratia rubefaciencs                                                                     64         58         61                                          FERM BP-1863                                                                  Enterobacter                                                                              21         28         29                                          aerogenes                                                                     ATCC 13048                                                                    Erwinia carotovora                                                                        165        188        174                                         FERM BP-1538                                                                  Citrobacter freundii                                                                      44         32         38                                          ATCC 8090                                                                     Corynebacterium                                                                           35         31         29                                          vitarumen                                                                     ATCC 10234                                                                    Hafnia alvei                                                                              40         35         38                                          ATCC 9760                                                                     Kluyvera citrophila                                                                       41         38         36                                          FERM P-3149                                                                   Salmonella  38         36         40                                          schottmuelleri                                                                ATCC 8759                                                                     Xanthomonas citri                                                                         47         43         41                                          FERM BP-1861                                                                  ______________________________________                                    

EXAMPLE 5

Escherichia Coli (ATCC 10798) was grown in the same medium as used inExample 2 at 37° C. for 16 hours in the same manner as in Example 2, 5ml of a previously sterilized 500mM phosphate buffer containing 200mM2',3'-dideoxyuridine and 200mM hypoxanthine was added to the aboveculture solution, and cultivation was continued for an additional 10hours. Measurement by means of high-performance liquid chromatographyshowed formation of 36 mg/dl of 2',3'-dideoxyinosine.

EXAMPLE 6

Washed cells of Escherichia coli (ATCC 10798) grown and treated in thesame way as in Example 2 were added to 10 ml of a 10mM phosphate buffer(pH 7.0) containing 100 mM 2',3'-dideoxyuridine and 100 mM adenine to aconcentration of 2%, and the mixture was incubated at temperatures shownin Table 4 for 24 hours. The concentration of 2',3'-dideoxyadenosineformed in each of the reaction mixtures was measured by means ofhigh-performance liquid chromatography. The results are shown in Table4.

                  TABLE 4                                                         ______________________________________                                        Reaction                                                                      temp. (°C.)                                                                       2',3'-Dideoxyadenosine formed (mg/dl)                              ______________________________________                                        40         611                                                                45         923                                                                50         1048                                                               55         673                                                                60         262                                                                ______________________________________                                    

EXAMPLE 7

Into shouldered 500 ml flasks was charged 50 ml each of a culture medium(pH =7.0) containing 500 mg/dl of a yeast extract, 1,000 mg/dl ofpeptone, 1,000 mg/dl of a meat extract and 500 mg/dl of NaCl, and theflasks were sterilized. One platinum loopful of Escherichia coli (ATCC10798), which had been cultured on a bouillon agar medium at 30° C. for16 hours, was inoculated in each medium contained in the flasks andcultured with shaking at 30° C. for 16 hours. The cells were separatedfrom the medium by centrifugation and washed with 0.05 M phosphatebuffer (pH=7.0). The cells were again separated by centrifugation toprepare washed cells.

The washed cells of Escherichia coil ATCC (10798) were added to 1 literof 1 mM phosphate buffer (pH=7.0) were added to containing 20 mM of DDUand 20 mM of Hyp, the concentration of the cells in said medium being 1%by weight. The reaction was allowed to proceed at 50° C. for 24 hours.As a result, there was produced 70 mg/dl of DDI (recovering rate=15%).

The cells were removed off by centrifugation (at 7,000 G for 40minutes), and then 50 mg of activated carbon (Shirasagi Charcoal,manufactured by Takeda Chemical Industries Co., Ltd.) was added afteradjustment to pH=8.0 by NaOH solution thereto. The resulting mixture wasmaintained at 50° C. for 1 hour, in order to remove off proteins and toeffect decoloration, and then filtered, using a filter having a poresize of 0.45 μm. The pH of the filtrate was adjusted to 8 by the use of1N NaOH, and the filtrate was then concentrated to 100 ml. The thusconcentrated solution was fed (at SV=1) to a column (having a diameterof 20 mm and a height of 210 mm) charged with 65 g of a porous nonpolaradsorption resin (SP 207, manufactured by Mitsubishi ChemicalIndustries, Ltd.) and then 300 ml of water was passed through the column(at SV=2). The thus obtained fraction is designated as Fraction I.Thereafter, 260 ml of aqueous 20% ethyl alcohol solution was passedthrough the column (at SV=2) to carry out elution. The fraction obtainedis designated as Fraction II. The above treatments with said resin werecarried out at a temperature of 30° C.

The fractions were analyzed by liquid chromatography. In Fraction I werecontained Ura and Hyp, which were recovered at a percentage of 99% and98%, respectively. In Fraction II were contained DDI and DDU, which wererecovered at a percentage of 98% and 95%, respectively.

The pH of Fraction II was adjusted to 8 by the addition of 1N NaOH, andit was then concentrated to crystallize DDI. There was obtained 410 mgof crystals of highly pure DDI. The results of elemental analysis of thethus obtained DDI are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Elemental Analysis                                                                     C          H       N                                                 ______________________________________                                        Calculated:                                                                              50.84%       5.12%   23.72%                                        Found:     50.86%       5.12%   23.91%                                        ______________________________________                                    

EXAMPLE 8

To a column having a diameter of 20 mm and a height of 210 mm andcharged with 65 ml of a porous nonpolar adsorption resin (SP 207,manufactured by Mitsubishi Chemical Industries, Ltd.) was fed (at SV=2)100 ml of concentrated solution containing 700 mg/dl of DDI prepared ina similar manner as in Example 1, and 250 ml of water was passed throughthe column (at SV=2) to obtain Fraction I'. Subsequently, 200 ml ofaqueous 10% isopropanol alcohol solution was passed through the column(at SV=2) to obtain Fraction II'. The treatments with said resin werecarried out at a temperature of 30° C.

The thus obtained fractions were analyzed by liquid chromatography.Fraction I' contained Ura and Hyp, which were recovered at a percentageof 99% and 97%, respectively. Fraction II' contained DDI and DDU, whichwere recovered at a percentage of 98% and 95%, respectively.

The pH of Fraction II' was adjusted to 8 by the addition of 1N NaOH, andthe fraction was then concentrated to crystallize DDI. There wasobtained 440 mg of crystals of highly pure DDI. The results of elementalanalysis of the thus obtained DDI are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Elemental Analysis                                                                     C          H       N                                                 ______________________________________                                        Calculated:                                                                              50.84%       5.12%   23.72%                                        Found:     50.87%       5.10%   23.81%                                        ______________________________________                                    

EXAMPLE 9

Into shouldered 500 ml flasks was charged 50 ml each of a culture medium(pH=7.0) containing 0.5 g/dl of a yeast extract, 1.0 g/dl of peptone,1.0 g/dl of a meat extract and 0.5 g/dl of NaCl, and the contents of theflasks were sterilized. One platinum loopful of Escherichia coli ATCC(10798), which had been cultured on a bouillon agar medium at 30° C. for16 hours, was inoculated in each medium contained in the flasks andcultured with shaking at 30° C. for 16 hours. The cells were separatedfrom the medium by centrifugation and washed with 0.05 M phosphatebuffer (pH=7.0). The cells were again separated by centrifugation toprepare washed cells.

The washed cells of Escherichia coli ATCC (10798) were added to 1 literof 100 mM phosphate buffer (pH=7.0) containing 20 mM of DDU and 20 mM ofAd, whereby the concentration of the cells in said medium was 1% byweight. The reaction was allowed to proceed at 50° C. for 24 hours. As aresult, there was produced 85 mg/dl of DDA (recovering rate=18%).

The cells were removed off by centrifugation (at 7,000 G for 40minutes), and then 50 mg of activated carbon (Shirasagi Charcoal,manufactured by Takeda Chemical Industries Co., Ltd.) was added afteradjusted to pH=8.0 by NaOH solution thereto. The resulting mixture wasmaintained at 50° C. for 1 hour, so as to remove off proteins and toeffect decoloration, and then filtered, using a filter having a poresize of 0.45 μm. The filtrate was concentrated to 15 ml and thenfiltered, using a No. 5 filter paper. Thirteen (13) grams of theconcentrated solution, which contained 6.2 g/dl of DDA, was fed at an SVof 1 to a column having a diameter of 20 mm and a height of 210 mm andcharged with a porous nonpolar synthetic adsorbing resin (SP 207,manufactured by Mitsubishi Chemical Industries, Ltd.), and then 260 mlof water was passed through the column at an SV of 2. The thus obtainedfraction is designated as Fraction I. Subsequently, 390 ml of aqueous10% ethyl alcohol solution was passed (at SV=2) to carry out elution.The thus obtained fraction is designated as Fraction II. Thereafter, 390ml of aqueous 20% ethyl alcohol solution was passed through the column(at SV=2) to effect additional elution. The thus obtained fraction isdesignated as Fraction III.

The fractions were analyzed by liquid chromatography. In Faction I wascontained U alone, the recovering rate of which was 99%. In Fraction IIwere contained Ad, DDU and a small quantity of DDA, the recovering ratesof Ad and DDU being 99% and 98,--respectively. In Fraction III wascontained DDA, the recovering rate of which was 95%.

Fraction III was concentrated to crystallize DDA. It was cooled to 10°C. and filtered to obtain 600 mg of highly pure DDA. The results ofelemental analysis of the thus obtained DDA are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Elemental Analysis                                                                     C     H          N       O                                           ______________________________________                                        Calculated:                                                                              51.06%  5.57%      29.77%                                                                              13.60%                                    Found:     51.22%  5.53%      29.78%                                                                              13.47%                                    ______________________________________                                    

EXAMPLE 10

To a column having a diameter of 20 nm and a height of 210 mm andcharged with 65 ml of a porous nonpolar synthetic adsorption resin (SP207, manufactured by Mitsubishi Chemical Industries, Ltd.) was fed (atSV=2) 13 ml of concentrated DDA-containing solution containing 6.2 g/dlof DDA prepared in a similar manner as in Example 1, and 260 ml of waterwas passed through the column (at SV=2)to obtain Fraction I'.Subsequently, 520 ml of aqueous 20% methyl alcohol solution was passedthrough the column (at SV=2) to carry out elution. The fraction obtainedwas designated as Fraction II'. Thereafter, 390 ml of aqueous 40% methylalcohol solution was passed (at SV=2) to effect additional-elution. Thethus obtained fraction is designated as Fraction III'.

The thus obtained fractions were analyzed by liquid chromatography. InFraction I' was contained U alone, the recovering rate of which was 98%.In Fraction II'were contained Ad, DDU and a small quantity of DDA, therecovering rates of Ad and DDU being 98% and 98%, respectively. InFraction III' was contained DDA, the recovering rate of which was 93%.

Fraction III' was concentrated to crystallize DDA. It was cooled to 10°C. and then filtered to obtain 580 mg of highly pure DDA. The results ofelemental analysis of the thus obtained DDA are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Elemental Analysis                                                                     C     H          N       O                                           ______________________________________                                        Calculated:                                                                              51.06%  5.57%      29.77%                                                                              13.60%                                    Found:     51.30%  5.53%      29.80%                                                                              13.37%                                    ______________________________________                                    

As described hereinabove, the process of the invention makes it possibleto separate and purify DDA in an effective manner by means of atreatment with a porous nonpolar resin. It can therefore be practicedcommercially.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A process for the purification of a2',3'-dideoxynucleoside from a solution obtained from a processrequiring a microorganism or an enzyme, said solution containing, asimpurities, other 2',3'-dideoxynucleosides or nucleic acid bases, saidprocess comprising:(i) contacting said solution with a porous, nonpolarresin to cause the adsorption of said 2',3'-dideoxynucleoside onto saidporous nonpolar resin, wherein said 2',3'-dideoxynucleoside is2',3'-dideoxyadenosine or 2',3'-dideoxyinosine; (ii) separating saidporous nonpolar resin from said solution; and (iii) fractionally elutingsaid adsorbed 2',3'-dideoxynucleoside to obtain a purified2',3'-dideoxynucleoside product.
 2. The process of claim 1, wherein saidporous nonpolar resin is a styrene-divinylbenzene resin.
 3. The processof claim 1, wherein said porous nonpolar resin is a modifiedstyrene-divinylbenzene resin.
 4. The process of claim 1, wherein said2',3'-dideoxynucleoside is 2',3'-dideoxyadenosine.
 5. The process ofclaim 4, wherein said porous nonpolar resin is a styrene-divinylbenzeneresin.
 6. The process of claim 4, wherein said porous nonpolar resin isa modified styrene-divinylbenzene resin.
 7. The process of claim 1,wherein said 2',3'-dideoxynucleoside is 2',3'-dideoxyinosine.
 8. Theprocess of claim 7, wherein said porous nonpolar resin is astyrene-divinylbenzene resin.
 9. The process of claim 7, wherein saidporous nonpolar resin is a modified styrene-divinylbenzene resin. 10.The process of claim 1, said solution containing, as impurities, saidother 2',3'-dideoxynucleosides and said nucleic acid bases.
 11. Theprocess of claim 4, said solution containing, as impurities, uracil and2',3'-dideoxyuridine.
 12. The process of claim 7, said solutioncontaining, as impurities, uracil and 2',3'-dideoxyuridine.
 13. Theprocess of claim 4, said solution containing, as impurities, adenine,uracil and 2',3'-dideoxyuridine.
 14. The process of claim 7, saidsolution containing, as impurities, uracil, 2',3'-dideoxyuridine andhypoxanthine.
 15. The process of claim 4, said solution containing, asimpurities, adenine.
 16. The process of claim 7, said solutioncontaining as impurities, hypoxanthine.